mmg_233_2014_genetics_genomicsfandomcom-20200215-history
Genetic Analysis: The Lac Operon
Overview: The Lac Operon is an operon present within E.Coli ''cells that codes for the synthesis of proteins needed to break down lactose into a usable energy source. This lac operon consists of many different elements such as: the lacI gene (codes for lacI repressor protein), the activator-binding site (where CAP binds), the promoter (where RNA polymerase binds), the operator (where the lacI repressor binds), the lacZ gene (encodes β-galactosidase), lacY gene (encodes lactose permease), and the lacA gene (encodes thiogalactoside transacetylase). ''E.Coli cells normally use glucose as their energy source because it produces much more energy than lactose, but in the absence of glucose the E.Coli ''cells can turn on the lac operon in order to switch over to the utilization of lactose as an energy source. Many genetic analysis experiments were done throughout the 1900’s to better understand the ability of this operon to be induced and the multiple regulatory mechanisms that give it the ability to function properly. Genetic Analysis of the Lac Operon: Key Scientists: François Jacob (17 June 1920 - 19 April 2013) and Jacques Monod (9 February 1910 – 31 May 1976) were French biologists who came up with the notion that control of enzymes levels within all cells occurs through regulation of transcription. They are famous for their work on the lac operon and in 1965 they shared the Nobel Prize in Medicine with André Lwoff. Enzymes Encoded by the Lac Operon Can Be Induced/Repressed: Early experiments were done in which ''E.coli ''was grown under different conditions to monitor the levels of the enzymes it produced. ''E.coli cells were first grown within medium containing just glucose and then in medium containing just lactose. When the cells were grown in the glucose medium, they didn’t produce the three enzymes (lacZ,Y,A) needed to break down lactose. On the other hand, when the cells were grown within the lactose medium, they DID produce the lacZ, Y, and A enzymes. This simple experiment provided the insight that there is some sort of “inducer” that causes E.Coli ''cells to produce the lacZ, Y, and A enzymes and some sort of “repressor” that causes ''E.''Coli cells to not produce the lacZ, Y, and A enzymes. Mutations in LacI gene Cause Constitutive Expression of Lac Operon: The next experiments done were more focused on the lacI gene and its contribution to the regulation of the lac operon. In this experiment, X-gal was used in order to measure the activity of β-galactosidase (lacZ). X-gal is a colorless analog of lactose that, when hydrolyzed by β-galactosidase, will turn blue and when not hydrolyzed by β-galactosidase will remain colorless. When wild ''E.Coli cells were grown on medium containing lactose as the major carbon source, the colonies were all blue, which proved that β-galactosidase production was being induced by the presence of lactose and therefore hydrolyzing the X-gal within the medium, turning it blue. When the wild E.Coli cells were grown on medium containing glucose as the major carbon source, the colonies remained white, which proved that β-galactosidase production was being repressed by the lack of lactose and therefore the X-gal wasn’t being hydrolyzed causing it to remain colorless. More interestingly, when the wild E.Coli ''cells were introduced to chemical mutagens before they were plated onto the glucose medium some blue colonies were produced. These mutated cells not only expressed all of the lac operon enzymes (lacZ,Y,A), but they expressed them at much higher levels than the wild-type cells. These cells were called “constitutive” because they failed to repress the lac operon and continuously expressed the lacZ, Y, and A enzymes. After further recombinational analysis, the mutations within these cells were mapped back to the lacI region of the operon, thus linking the ability of the lac operon to be repressed to the lacI region. Further Hypotheses and Grounds for More Genetic Analysis: Jacob and Monod hypothesized that the mutants generated from the above experiment had a defect within a protein that would normally repress the lac operon in the absence of lactose. They fittingly named this protein the lac repressor and further hypothesized that it bound to a region on the operon where transcription was initiated and therefore blocked the process of transcription in the absence of lactose. In addition to these hypotheses, they hypothesized that when lactose was present within the ''E.Coli cells it bound to the lac repressor and inhibited its ability to bind to the DNA strand, which prevented it from repressing transcription. This would lead to the transcription of the lac operon and the expression of the lac enzymes (lacZ,Y,A). These simple genetic analysis experiments are what lead to the vast understanding and appreciation of the lac operon of E.Coli cells within today's society. Resources: Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 10.1, Bacterial Gene Control: The Jacob-Monod Model. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21683/ Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000. Discovery of the lac system: negative control. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21954/ François Jacob on Wikipedia.org Jacques Lucien Monod on Wikipedia.org